A Pyruvate-dependent One-carbon Metabolic Pathway For The Assimilation Of Formaldehyde And Methanol To Synthesize 1,3-propanediol In Escherichia Coli

Formaldehyde(HCHO)is an important intermediate in the metabolism of one-carbon(C1)compounds such as methanol,formate and methane.These C1 compounds can all be converted to formaldehyde and then used to supply the energy and substances required for cell growth through central metabolic pathways.Currently,the ribulose monophosphate(RuMP)cycle is the mostly studied pathway for the assimilation of methanol and formaldehyde.However,causing the regeneration of Ru5P is not efficient in the RuMP pathway of synthetic methylotrophy,which limits the utilization of methanol and formaldehyde.In this work,in order to achieve the utilization of methanol and formaldehyde without using the RuMP pathway,a pyruvate-dependent 2-keto-4-hydroxybutyrate aldolase was screened and selected for formaldehyde fixation.The aldolase was then combined with other three enzymes to construct a pyruvate-dependent C1 metabolic pathway for the production of 1,3-propanediol(1,3-PDO).Compared with other synthesis pathways of 1,3-PDO,this pathway has the following advantages:1)C1 carbon is directly channeled into a precursor of 1,3-PDO,opening a new biosynthetic route with a higher atom efficiency of substrate utilization;2)the use of pyruvate as an acceptor for HCHO is glycerol-independent,circumventing thus the need of coenzyme B12 as cofactor for glycerol dehydration;3)the pathway is much shorter and more simple than the recently proposed homoserine-dependent 1,3-PDO pathway,avoiding thus complicated regulations.Additionally,after in vivo and in vitro studies and optimization of this new 1,3-PDO pathway,the 1,3-PDO titer was significantly increased.The main results of this thesis are summarized as follows.1.Excavating the potential aldolase for formaldehyde fixation:in order to avoid the problems involved in the assimilation of formaldehyde via the RuMP pathway,we screened and selected a Class I pyruvate-dependent 2-keto-4-hydroxybutyrate aldolase(KHB)with a high activivty that catalyzes the formation of 2-keto-4-hy droxybutyrate from formaldehyde and pyruvate.It plays an important role in the fixation of formaldehy.In addition,the KHB enzyme was involved into the novel biosynthesis pathway of 1,3-PDO.2.Biosynthesis of 1,3-PDO from formaldehyde and pyruvate in vitro:to confirm whether the newly proposed pathway can be used for the production 1,3-PDO,we first examined it in vitro.It was confirmed that this pathway can use formaldehyde and pyruvate as substrates for the production of 1,3-PDO(with 35.3±0.4 mg/L 1,3-PDO formed without optimization).According to the activity assay of the branched-chain alpha-keto acid decarboxylase(KDC)in vitro,there is a relationship between KDC and the formation of by-products lactate and ethanol.3.Construction of a novel pyruvate-based C1 metabolic pathway for the production of 1,3-PDO in vivo:In order to achieve the in vivo production of 1,3-PDO from methanol or formaldehyde,we used multi-gene co-expression strategies and constructed a pyruvate-dependent C1 metabolic pathway for the production of 1,3-PDO in E.coli.Through the analysis of protein expression,it was found that the protein expression in the pathway was relatively good,which provided a material basis for subsequent research.4.Production of 1,3-PDO from formaldehyde or methanol and glucose in vivo:using formaldehyde and glucose as co-substrates in batch and fed-batch fermentation with the strain BP3,we obtained 30±1.5 mg/L and 298.3±11.4 mg/L 1,3-PDO respectively.We further examined whether this pathway can produce 1,3-PDO using methanol and glucose in E.coli in vivo.Using fed-batch fermentation with strain BP4,3.8 mg/L 1,3-PDO were obtained.5.Optimization of the novel metabolic pathway:in order to decrease the transformation of formaldehyde to formate,the strains BP3?frmA and BP4?frmA were constructed by deleting the formaldehyde dehydrogenase A gene(frame)using the CRISPR-Cas9 system.Fed-batch fermentations were carried out,and the yield of 1,3-PDO was increased by 70.4%(508.3 ± 9.1 mg/L vs.298.3±11.4 mg/L)and 760%(32.7±0.8 mg/L vs.3.8 mg/L)respectively.It was confirmed that by knocking out the format gene,it was indeed possible to increase the yield of 1,3-PDO.